Azo dye degrading bacterium RL3 and application thereof

Abstract

The invention discloses an azo dye degrading bacterium RL3 and application thereof, and belongs to the technical field of microorganisms. According to the present invention, the azo dye degradation yeast RL3 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is July 25, 2023, and the preservation number is CGMCC NO.28008; the strain can decolorize azo dyes, especially reactive brilliant red K-2BP at room temperature, the decolorization rate within 16 h can reach 99% or above, the effect is remarkable, and a new technical thought is provided for treatment of dye wastewater or dye contaminated soil.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to an azo dye degrading bacterium RL3 and its applications. Background Technology

[0002] The dye industry is one of the most environmentally polluting sectors within the chemical industry. Dye wastewater is characterized by high color intensity, high organic matter concentration, complex composition, and a high content of recalcitrant substances, including large amounts of inorganic salts and sulfides, making it difficult to treat. Azo dyes, a type of synthetic dye named for the azo group in their molecular structure, are widely used in textiles, dyeing, papermaking, and printing. Due to their complex aromatic hydrocarbon molecular structure, azo dyes are stable, highly resistant to light and oxidation, and can persist in aquatic environments for extended periods, thus causing damage to aquatic ecosystems.

[0003] Common methods for treating azo dye wastewater include physicochemical methods and biological methods. Currently used physicochemical methods include adsorption decolorization, coagulation and sedimentation, chemical oxidation, ion exchange, ultrafiltration membrane decolorization, photocatalysis, and high-voltage pulse electrolysis. Compared to physicochemical methods, biological methods are more economically feasible and environmentally friendly, making them an important approach to water pollution control and attracting significant attention in the treatment of azo dye wastewater.

[0004] Traditional biological wastewater treatment methods, such as the activated sludge process, are low-cost and produce minimal secondary pollution. However, due to the difficulty in biodegrading azo dyes, the decolorization effect of ordinary activated sludge microorganisms is not ideal, limiting its application. In recent years, researchers have obtained various bacterial strains capable of degrading azo dyes from sludge or soil from dye wastewater treatment plants. These bacteria are diverse, including anaerobic, aerobic, and facultative anaerobic bacteria, with Pseudomonas and Bacillus being representative examples. The reductive degradation of azo dyes by bacteria is mainly completed under anaerobic conditions by azo reductases. However, the anaerobic degradation products of azo dyes are mostly toxic aromatic amines, which have carcinogenic, teratogenic, and mutagenic effects, easily accumulating in the environment and posing a threat to various organisms and human health.

[0005] Besides bacteria, filamentous fungi and yeasts also have good decolorizing effects on azo dyes. Fungal decolorization mechanisms are divided into adsorption decolorization and degradation decolorization. Most fungi, including filamentous fungi, first adsorb and then degrade, while some only adsorb. Many reported fungi have shown certain advantages in terms of the broad spectrum of adsorption and decolorization of dyes, mineralization rate, and decolorization speed.

[0006] Yeasts, as a large group of single-celled fungi, possess advantages such as rapid growth and metabolism, the ability to produce various fungal enzymes, ease of reactor design, and high wastewater treatment efficiency. They are also more tolerant of the high organic load and high salinity of dye wastewater, making them highly promising for wastewater treatment. Some researchers have analyzed the metabolites of azo dyes by salt-tolerant yeasts, demonstrating that their biodegradation process does not produce harmful aromatic amines, showcasing their potential value in treating azo dye wastewater. Therefore, developing more azo dye-degrading bacterial strains to achieve safe and environmentally friendly decolorization of azo dyes and improving their decolorization efficiency are urgent problems that need to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide an azo dye degrading bacterium RL3 and its applications, in order to solve the problems existing in the prior art.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] This invention provides an azo dye-degrading yeast, Kurtzmaniella quercitrusa RL3, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 25, 2023, with accession number CGMCCNo. 28008.

[0010] The present invention also provides the application of the above-mentioned azo dye degrading yeast RL3 in the preparation of azo dye decolorizing agents.

[0011] Preferably, the azo dye comprises Reactive Brilliant Red K-2BP.

[0012] The present invention also provides an azo dye decolorizing agent, wherein the azo dye decolorizing agent contains the above-mentioned azo dye degrading yeast RL3.

[0013] The present invention also provides the application of the above-mentioned azo dye degrading yeast RL3 or the above-mentioned azo dye decolorizing agent in the treatment of pollutants containing azo dyes.

[0014] In this invention, the pollutants include, but are not limited to, dye wastewater or soil contaminated by dye wastewater.

[0015] Preferably, the azo dye comprises Reactive Brilliant Red K-2BP.

[0016] The present invention also provides a method for treating wastewater containing azo dyes, comprising the step of inoculating the above-mentioned azo dye-degrading yeast RL3 into the wastewater containing azo dyes and culturing it to degrade the azo dyes in the wastewater.

[0017] Preferably, the inoculum amount of the azo dye-degrading yeast RL3 is 4% to 6%, more preferably 5%.

[0018] Preferably, the culture temperature is 20-30°C, more preferably 25°C; and the culture time is 16 hours or more.

[0019] Preferably, the azo dye comprises Reactive Brilliant Red K-2BP.

[0020] The present invention discloses the following technical effects:

[0021] This invention isolates a yeast strain RL3 from soil, biologically classified as *Kurtzmaniella quercitrusa*. Previous literature reports that strains of this type have potential applications in biotransformation processes; they exhibit efficient utilization of pentoses and growth potential under low pH conditions, making them promising candidate strains for industrial production. In this invention, this strain can decolorize azo dyes, especially Reactive Brilliant Red K-2BP, at room temperature, achieving a decolorization rate of over 99% within 16 hours, demonstrating significant effectiveness. This provides a new technical approach for the treatment of dye wastewater or dye-contaminated soil, laying a technological foundation for the safe and environmentally friendly treatment of azo dye pollutants and for resource recycling. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a diagram showing the growth of strain RL3 after isolation and purification.

[0024] Figure 2 This is a microscopic image of the bacterial cell morphology of strain RL3.

[0025] Figure 3 Phylogenetic tree of strain RL3;

[0026] Figure 4 The results of shake-flask decolorization of strain RL3 are shown in the image; the left flask is the shake flask inoculated with strain RL3, and the right flask is the shake flask without strain RL3.

[0027] Figure 5 Image showing the decolorization effect of strain RL3 in an SBR reactor. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1: Isolation, Activation, and Preservation of Microbial Strains

[0034] 1. Isolation and purification of dye-degrading strain RL3

[0035] This azo dye-degrading bacterium was obtained from the soil of the fourth flower bed in the East Campus of the University of Chinese Academy of Sciences (Yanqi Lake Campus) through dilution, spreading, and streak isolation. The culture conditions were 28–30℃ for 7 days.

[0036] 1) The specific procedures for dilution and spreading of dye-degrading bacteria RL3 are as follows: Weigh 0.1g of soil sample and add it to a test tube containing 9mL of water. Mix well and let stand for separation. During separation, in a clean bench, use a pipette to aspirate 100μL of soil suspension and add it to the dye-selective medium. Spread evenly using a disposable sterile spreading stick. Incubate at 30℃ for 7 days to observe colony growth (instantly incubate the medium upright until the surface is slightly dry, then invert the medium for further incubation).

[0037] 2) Streak isolation of dye-degrading bacteria RL3: Pick a single colony with a degradation zone on the dye selective medium with an inoculation loop, streak it on a new dye selective medium, and isolate and purify the colony. Figure 1 The culture medium after two streak separations shows that the isolated single colonies are round, smooth, raised, and moist, with a diameter of 1-2 mm, and are milky white (pink due to dye enrichment). A transparent degradation zone forms around the colony.

[0038] 3) Microscopic observation of dye-degrading bacteria RL3: The morphology of the bacteria was observed under a 10x eyepiece and a 40x objective lens using a water slide (see...). Figure 2 As can be seen, the bacterial cells are oval-shaped, colorless and transparent, and have spore structures.

[0039] The formula for the dye-selective culture medium is as follows: glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, ammonium chloride 1 g / L, magnesium sulfate 0.5 g / L, yeast extract 0.2 g / L, agar 20 g, and Reactive Brilliant Red (K-2BP) 50 mg / L (stock solution 1000 mg / L). Sterilization conditions are 115℃ for 30 min. When the culture medium temperature drops to 50–60℃, pour 15–20 ml of the medium into each disposable sterile petri dish and cool to room temperature to complete the preparation.

[0040] 2. Activation of the strain

[0041] After streaking the culture twice or more, pick up a loopful of a single colony from the dye-selective medium and inoculate it into a liquid medium containing 10 mg / L Vitality Brilliant Red. Incubate at 30°C and 180 rpm until the culture medium turns milky white.

[0042] The formulation of the liquid culture medium used for activation was as follows: glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, ammonium chloride 1 g / L, magnesium sulfate 0.5 g / L, yeast extract 0.2 g / L, and Reactive Brilliant Red (K-2BP) 10 mg / L (the stock solution was 1000 mg / L). The sterilization conditions were 115℃ for 30 min.

[0043] 3. Preservation of bacterial strains

[0044] Prepare a 50% (v / v) glycerol solution and sterilize it (115℃, 30 min). Mix the reactivated bacterial solution and the sterilized glycerol in a 1:1 (v / v) ratio in a cryovial. Store at -80℃.

[0045] 4. Molecular identification of the strain

[0046] After extraction and PCR amplification of the RL3 gene, ITS sequencing was performed to construct a phylogenetic tree (see...). Figure 3Genomic DNA was extracted and amplified by PCR using universal primers ITS1 (SEQ ID NO.1: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO.2: 5'-TCCTCCGCTTATTGATATGC-3'). PCR amplification consisted of three stages under the following conditions: Stage 1: 94℃ pre-denaturation for 5 min, one cycle; Stage 2: 94℃ denaturation for 30 s, 50℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 35 cycles; Stage 3: 72℃ extension for 7 min, one cycle, PCR completed. The amplified DNA was then sequenced and stored at 4℃. Sequencing was performed using the Sanger sequencer, an ABI 3730x. The sequenced DNA is shown in SEQ ID NO.3.

[0047] SEQ ID NO.3:

[0048] .

[0049] The sequencing results were compared with the ITS sequence homology using online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) on NCBI (https: / / www.ncbi.nlm.nih.gov / ). The strain sequence showed 99.34% homology with *Kurtzmaniella quercitrusa* strain, and it was classified and named *Kurtzmaniella quercitrusa* RL3. It was deposited on July 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO.28008. Testing on July 25, 2023, confirmed the viability of the bacterial cells.

[0050] Example 2: Decolorization verification of strain RL3

[0051] 1. Shake-flask decolorization experiment

[0052] After the dye-degrading strain RL3 was isolated by streak stripping more than twice, a single colony was picked from one loop of culture medium with an inoculation loop and inoculated into liquid culture medium containing 50 mg / L Reactive Brilliant Red (K-2BP). The culture was carried out at 30°C and 160 rpm for 7 days. Two replicates were made, namely RL3-1 and RL3-2. The sterilized liquid culture medium containing Reactive Brilliant Red without inoculation of strain RL3 was used as a control.

[0053] The formulation of the sterile liquid culture medium for Reactive Brilliant Red (K-2BP) is as follows: glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, ammonium chloride 1 g / L, magnesium sulfate 0.5 g / L, yeast extract 0.2 g / L, and Reactive Brilliant Red (K-2BP) 50 mg / L (stock solution 1000 mg / L). The sterilization conditions are 115℃ for 30 min.

[0054] 2. Decolorization experiment in SBR reactor

[0055] The sequencing batch reactor activated sludge process (SBR) is a wastewater treatment method that uses activated sludge and consists of five basic steps in the same reactor, arranged sequentially over time: influent, aeration, sedimentation, effluent discharge, and standby. This experiment used an SBR reactor, replacing the activated sludge with strain RL3. Influent, aeration, and effluent discharge were sequentially performed in the reactor to verify the decolorization and degradation effect of the test strain RL3 on the dye liquor.

[0056] The SBR reactor used has a volume of 2L. A 1L dye solution to be decolorized was prepared with the following formula: glucose 10g / L, potassium dihydrogen phosphate 1g / L, ammonium chloride 1g / L, magnesium sulfate 0.5g / L, yeast extract 0.2g / L, and Reactive Brilliant Red (K-2BP) 20mg / L (the mother liquor was 1000mg / L). No sterilization was required; the solution was directly introduced into the SBR reactor.

[0057] RL3 activated with the azo dye Reactive Brilliant Red (OD value reaching 1.3–1.5) was added to the SBR reactor at 5% (v / v). The aeration flow rate was 0.5 NL / min to maintain dissolved oxygen (DO) at 4–5 mg / L, and the reactor was run at room temperature (25°C) for 16 h.

[0058] 3. Colorimetric determination and decolorization rate calculation

[0059] Colorimetric detection was performed using a UV spectrophotometer to measure the absorbance of the supernatant at the dye's maximum absorption peak. Reactive Brilliant Red (K-2BP) has a maximum absorption peak at λ = 534 nm. After centrifuging the decolorized solution at 8000 rpm for 3 min, the absorbance (OD) of the supernatant was measured at λ = 534 nm. Pure water was used as a blank for zeroing. The decolorization rate was calculated using the following formula:

[0060] Decolorization rate (%) = (AB) / A*100, where: A is the OD value of the test liquid before decolorization, and B is the OD value of the supernatant after centrifugation after decolorization.

[0061] 4. Results

[0062] The decolorization effect of the shake-flask experiment (two parallel experiments: RL3-1 and RL3-2) is shown in [reference needed]. Figure 4 The decolorization rate is shown in Table 1. After degradation by RL3, the decolorization effect is significant, the culture medium is milky white, and the decolorization rate can reach more than 91%.

[0063] The decolorization effect of the SBR reactor experiment is shown in [reference needed]. Figure 5 The decolorization rate calculation results are shown in Table 2. After degradation by RL3, the decolorization effect is significant, with a decolorization rate of over 99%.

[0064] Table 1. Dye Degradation and Decolorization in Shake Flask Experiments (Culturing at 30℃ and 160rpm for 7 days)

[0065] Experimental group absorbance Decolorization rate RL3-1 0.037 91.70％ RL3-2 0.012 97.00％ CK 1.065 8.62％

[0066] Table 2. Dye Degradation and Decolorization in SBR Reactor Experiment (Running at 25℃ for 16 hours)

[0067] Experimental group absorbance Decolorization rate SBR inlet 0.428 / SBR effluent 0.004 99.07％

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An azo dye-degrading yeast Kurtzmaniella quercitrusa RL3, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of July 25, 2023 and a deposit number of CGMCC No.28008.

2. Use of the azo dye degrading yeast RL3 according to claim 1 in the preparation of an azo dye decolorizing agent.

3. The use according to claim 2, characterized in that: The azo dye includes Reactive Brilliant Red K-2BP.

4. An azo dye decolorizing agent, characterized in that The azo dye decolorizing agent comprises the azo dye degrading yeast RL3 according to claim 1.

5. Use of the azo dye degrading yeast RL3 according to claim 1 or the azo dye decolorizing agent according to claim 4 in treating pollutants containing azo dyes.

6. The use according to claim 5, characterized in that: The azo dye includes Reactive Brilliant Red K-2BP.

7. A method for treating wastewater containing azo dyes, characterized in that: The method comprises the steps of inoculating the azo dye degrading yeast RL3 described in claim 1 into wastewater containing azo dyes for culturing so as to degrade the azo dyes in the wastewater.

8. The method according to claim 7, characterized in that The inoculation amount of the azo dye degrading yeast RL3 is 4% to 6%.

9. The method according to claim 7, characterized in that: The culture temperature is 20-30° C., and the culture time is more than 16 hours.

10. The method according to any one of claims 7 to 9, characterized in that: The azo dye includes Reactive Brilliant Red K-2BP.

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